Page 94 - GTM-2-4

Global Translational Medicine ECM receptor pathway in endotheliocytes after MI

microbeads. A portion of the isolated cells were labeled in the mobile phase A of a liquid chromatography system
with CD31-PE, followed by a flow cytometric examination and separated using an EASY-nLC1000 ultra-performance
to determine the concentration of ECs. The remaining cells liquid chromatography system.
were cryogenically preserved in a liquid-nitrogen bath and The separated polypeptides were ionized via an NSI
subsequently transferred to an −80℃ storage unit. ion source and were subsequently analyzed using Orbitrap

2.2. Tandem mass tag-proteome sample Fusion mass spectrometry. The ion source voltage was set
preparation, high-performance liquid at 2.0 kV, facilitating the detection and analysis of both the
chromatography sorting, and mass spectrometry parent ion and secondary fragments of the polypeptides by
detection a high-resolution Orbitrap. The scan range was set from
400 – 1500 m/z, and the resolution was set to 60000. For
Protein lysate containing 1% TritonX-100 and 1% protease secondary mass spectrometry, the scanning range was set
inhibitor was added to the sample. Following a 4-h to 100 m/z, with a resolution of 15000. Data acquisition
incubation, the mixture was centrifuged at 12,000 rpm was performed using data-dependent acquisition settings,
for 10 min, after which the cell debris was removed. The which sequentially scanned the top 20 peptide parent ions
supernatant, harboring the extracted protein, was then with the most substantial signal intensity into a higher-
transferred to a new Eppendorf tube, and the protein energy collisional dissociation collision cell pool. The
concentration was quantified using a Bicinchoninic fragmentation energy was set to 32%, and secondary mass
acid protein assay kit. Based on the concentration spectrometry was carried out sequentially. To enhance
determination, equivalent protein amounts were selected, mass spectrometry (MS) utilization, automatic gain control
and the sample volume was adjusted to 500 µL with 8M
urea. Subsequently, a 20% trichloroacetic acid (TCA) was was set at 5e4, the signal threshold was set at 20000 ions/s,
added and incubated in ice water for 2 h or overnight. the maximum injection time was set at 70 ms, and dynamic
After centrifugation at 4℃ for 5 min, the supernatant was exclusion was employed for 30 s to prevent redundant
discarded, and the pellet was washed three times with scanning of parent ions.
ice-cold acetone. The protein precipitate was air-dried, 2.3. Data analysis
followed by the addition of 200 mm tetraethylammonium
bromide (TEAB) and dispersion by ultrasound. Trypsin, Infarct size, determined through TTC staining, and
at a ratio of 1:50 (protease: protein, M/M), was then added collagen fiber area, assessed through Sirius Red staining,
for overnight hydrolysis at 37℃. Following this enzymatic was quantified using ImageJ. Statistical analyses were
digestion, 5 mm dithiothreitol (DTT) was added to achieve performed using Prism 7.0, and statistical significance
a final concentration and reduced at 37℃ for 1 hour. A final was determined through Student’s t-test. The resulting
concentration of 15 mm iodoacetamide (IAM) was added data were presented with a 95% confidence interval, where
and left in the dark at room temperature for 45 min. P < 0.05 signified statistical significance.
The trypsin-hydrolyzed peptides were then desalted Secondary MS data were acquired using Maxquant
with Strata X C18 (Phenomenex) and lyophilized. The (v1.5.2.8) with the following parameters: the Mus_
peptides were reconstituted with 0.5 M TEAB and labeled musulus_10090_SP_20191115 (17,032 sequences) was
according to the instructions provided by the tandem mass employed, supplemented with a reverse library for
tagging (TMT) kit (label reagents for sham-1, sham-2, false positive rate calculation due to random matching.
sham-3, MI-1, MI-2, and MI-3 were 126, 127, 128, 129, A common contaminated library was included to
130, and 131, respectively). The labeling procedure eliminate the potential impacts of protein contamination.
involved dissolving the thawed reagents in acetonitrile, The digestion mode employed trypsin/P was the digestive
mixing them with the peptides, incubating them at room mode, allowing for a maximum of 2 missing cleavage
temperature for 2 h, and subsequent desalting and freeze- sites and a minimum peptide length of 7 amino acids.
drying. Mass error tolerance rates for primary and secondary
ions in the first and main searches were set at 20 ppm
Next, the peptides were fractionated using high-pH
reverse-phase high-performance liquid chromatography and 5 ppm, respectively, with the secondary fragment
ion set at 0.02 Da.
(HPLC) on an Agilent 300 Extend C18 column (5 µm
diameter, 6 mm inner diameter, 250 mm length). Elution The fixed modification involved the alkylation of
occurred with a gradient of 8 – 32% acetonitrile, pH 9, over cysteine, and variable modifications included methionine
60 min, resulting in 60 fractions that were subsequently oxidation, N-terminal acetylation, and deamidation
combined into 14, followed by lyophilization for the (NQ). The quantification method was conducted using
subsequent steps. Finally, the polypeptides were dissolved a TMT-6plex kit, and both protein identification and

Volume 2 Issue 4 (2023) 3 https://doi.org/10.36922/gtm.2217